PROJECT SUMMARY Small-animal models are powerful discovery tools in medical research, but sacrificing prevents long-term, in vivo observation of natural or pathological processes. As such, there is a need for a morphologic, functional, cellular/molecular, and quantitative imaging technique capable of longitudinal visualization of biochemical and pharmacological processes in small-animal disease models. Unfortunately, current molecular optical imaging approaches tend to present an undesirable trade-off between imaging depth and resolution. Non-invasive photoacoustic imaging (PAI), which is capable of simultaneous anatomical, functional, and molecular visualization of pathology with high contrast/resolution at depth, has thus generated significant excitement among preclinical imaging researchers. However, these end-users currently lack a reliable, reproducible, and validated molecular PAI platform to complement their translational research. To address this need, we propose enabling the molecular sensitivity of PAI through the development and validation of targeted contrast agents and signal/image processing algorithms to allow simultaneous, reproducible, quantitative, longitudinal, and tomographic imaging of molecular and physiological signatures of disease and therapy response in preclinical studies. Many available molecular contrast agents lack adequate PAI contrast for deep imaging and/or overlap with spectral features of hemoglobin absorption, making it difficult to differentiate a targeted probe from surrounding blood. To address these limitations, we seek to continue development of a unique contrast agent based on antibody-targeted liposomes loaded with J-aggregates of indocyanine green (ICG) dye. Encapsulation of ICG J-aggregates in a liposomal compartment results in a stable contrast agent (Lipo-JICG), which provides highly advantageous properties for in vivo PAI: (i) a strong, narrow absorbance at ~890 nm, where it can be readily unmixed from hemoglobin spectra; (ii) enhancement of PAI signal due to dye-aggregation-mediated increases in thermal gradients and absorbance; (iii) the ability to implement robust, semi-quantitative PAI analysis that does not interfere with imaging of important physiological parameters such as blood oxygen saturation. Our compelling preliminary data show that targeted Lipo-JICG provides impressive stability, linearity, PAI-signal intensity and molecular specificity. During this research proposal, we will validate the molecular- imaging capabilities of this promising technology in tissue-mimicking phantoms, well-characterized cell cultures, and orthotopic models of ovarian cancer. At the conclusion of these studies, we will be in position to start mass- production and end-user dissemination of Lipo-JICG and image processing algorithms as a fully validated, molecularly specific PAI platform for reliable, reproducible, and affordable preclinical imaging. Although not the principle objective of this proposal, these studies also provide a foundation for clinical translation of our agent as the liposomes, ICG, and humanized-targeted antibodies of which it is composed have all been FDA cleared for i.v. use, therefore reducing safety concerns and improving the chances for future clinical utilization.